Folic acid production



'volved in the peptide linkages.

Patented Mar. 10, 1953 'lnazoo, Mich, assignors to The Upjohn Company, Kalamazoo, Mich., a corporation of Michigan NoDrawing. Application December 3,1948,

Serial No. 63,453

:1 This invention relates to a method for the preparation of N-'( (2 amino '4 hydroxy 6 pteridyDmethyl) -'N- (arylsulfonyl) p aminob'enzoate compounds and to certain intermediates useful in the method.

The N (2-amino-4-hydroxy-6-pteridyl) methyl) -'N -'(arylsulfonyl) p -aminobe'nzoate compounds which can be prepared'bythe method'of the invention have the generic formula aryl-S g wherein R isfrom the group consisting of hydrogen and the alkyl radicals and n is from the group consisting of zero and the positive integers 1 to 7., inclusive, and thus-include N-substitution products of p-aminobenzoic acid, of p-aminobenzoyl glutamic acid, of the p-aminobenzoylglutamyl-glutamic acids having up to seven glutamic acid residues in the molecule, and of their esters. In the structural'formulae given herein and in the appended claims aromatic nuclei are represented by one or more simple hexagons. In the naming of compounds having the generic formula given, which are described and claimed in copendi'ng application'Serial No. 41, 882, filed July 31,1948, and of other starting compounds and intermediate compounds'mentionedherein wherein both a glutamic. acid residue and a .peaminoben'zoi'c acid residue are included in the molecule, the nitrogen atom of the glutamic acid residue is,.'ior convenience, herein referred to by the symbol N' and the nitrogen atom of the p-aminobenzoic' acid residue is referred toby'the symbol N. As indicatedby the generic formula, compounds used in or prepared by the process of the invention which contain more than one glutamic acid or esterresidue contemplated by the invention are those wherein only the gamma-carboxyl-groups are in- Preferred compounds used in or prepared by the method of the invention are those wherein n'of the generic 'formula given, and in those given subsequently,

represents zero or the integer 1,"i. e.th'ose containing either-no glutamic acid or'ester residue or only one glutamic acid "or ester residue, and 50 aikyl-o-non-cnon-cm-n reclaims, (01. 260-25115) 2 the method and intermediate compounds will be described with particular reference thereto.

Compounds having the generic formula given above and compounds useful as intermediates in the preparation thereof are of particular value due to the relationship of the former with, and to theease with which they can be converted to, compounds similar to or identical with certain naturally occurring compounds of the folic acid group. Thus, diethyl N'-(N-((2-amino -5lhydroxy-G-pteridyl) methyl) N (p-toluenesulfonyl) -'p-aminobenzoyl) -1--glutamate can be converted readily by hydrolysis of the two ethyl ester groups with alkali to the corresponding vdicarboxy compoundand thelatter, by splitting the p- -toluenesulfonyl radical from the molecule with hydrogenbromide and a bromineacceptor in an aliphatic acid medium, according to the method described-and claimed in copending application,

-SerialfNo. 41,883, filed July 31, 1948, can be condues having the :same configuration in folic acid compounds isolated from natural sources. The method of the invention and the intermediate acid residues involved.

2,3-oxidopropanal dlalkyl acetal C O 0-a1k 1 H-N C O.(NH( J:HCHzCHzCO),O-a1kyl ary1- Oi 11 v 'N-(arylsulionyl)-p-aminobenzoate ester followed by l mine i ii drol sis when rcatalvyst) .fr ee ant isde'sired) 0 0 O R CO'(NH(5HCH5CH1C 0),.012

aryl O2 7 III 'N-(3,3-dialkoxy-2=hydroxypropy1)*N-(arylsulfonyl)-p -aminobenzoatecompound N-(3,3-dialkoxy-2-ketopropyl)-N-(arylsulionyl)-p-aminobenzoate compound 2,4,5'trlamino-G- hydroxypyrimidine NHi ' an N- (3,3-dialkoxy-Z-hydroxypropyl) -I I- (arylsulfonyl) -p-aminobenzoate compound (III) e. g. with chromic oxide. N-(3,3-dialkoxy-2-hydroxypropyl) N- (arylsulfonyl) -p-aminobenzoate compounds (III) which are esters, i. e. wherein R. is an alkyl radical, are prepared in convenient 'fashion by reacting a 2,3-oxidopropanal dialkyl acetal (I) with an N-(arylsulfonyll-p-aminobenzoate ester (II). The N-(3,3-dialkoxy-2-hydroxypropyl) -N- (arylsulfonyl) -p-aminobenzoate compounds (III) and the N-(3,3-dialkoxy-2- ketopropyl) -N-(arylsulfonyl) p aminobenzoate compounds (IV) which are esters, i. e. compounds wherein R' is an alkyl radical, can be converted under carefully controlled conditions to the corresponding acids, i. e. to compounds wherein R is hydrogen, by hydrolysis with alkalies and subsequent acidification.

Certain of the N-(arylsulfonyl)-p-aminobenzoate esters (II) are described and claimed in copending application Serial No. 41,888, filed July 31, 1948. They can be prepared readily, as described in the copending application just mentioned, by reacting an arylsulfonyl halide with an alkyl ester of p-aminobenzoic acid, p-aminobenzoyl-glutamic acid, or a p-aminobenzoylglutamyl-glutamic acid having up to '7 glutamic acid residues in the molecule.

Dialkyl acetals of 1,2-oxidopropanal which can be used in the process include the dimethyl, di-

ethyl, di-n-propyl, di-iso-propyl, dibutyl, diamyl and other dialkyl acetals. As a matter of convenience and availability dialkyl acetals wherein the alkyl radicals each contain less than 8 carbon atoms are preferred, although others can be used, if desired. It is apparent that the nature of the alkyl groups in the 3,3-dialkyl-Z-hydroxypropyl and in the 3,3-dialkyl-2-ketopropyl radicals of the intermediate compounds (III) and (IV) are determined by the particular 1,2-oxidopropanal dialkyl acetal used in the first step of the process.

Esters which can be used as starting compounds in the process, with the production of the corresponding ester intermediates, include the methyl, ethyl, n-propyl, iso-propyl, butyl, amyl, hexyl, nonyl and other alkyl esters. As a matter of convenience, alkyl esters containing less than about 8 carbon atoms in the alkyl ester radicals are preferably used in the process, although insofar as is known any alkyl ester can be used.

Although starting compounds containing substantially any arylsulfonyl radical can be used in the process of the invention and in the preparation of the corresponding arylsulfonyl intermediates and final products, the preferred starting materials and intermediate products are those containing the p-toluenesulfonyl radical due to the ready availability of the p-toluenesulfonyl halides and to the generally crystalline nature of the p-toluenesulionyl derivatives of compounds with which the present invention is concerned. Furthermore, it appears that the p-toluenesulionyl radical can frequently be removed from the compounds involved herein, by splitting with hydrogen bromide as mentioned previously, somewhat more readily and with the formation of a smaller proportion of undesired by-products than is the case with some of the other arylsulfonyl radicals. The invention is, however, not limited to compounds containing the p-toluenesulionyl radical and compounds containing other arylsulfonyl radicals, such as the o-toluenesulfonyl, naphthalenesulfonyl, methylnaphthalenesulfonyl radicals and others, can be used, if desired.

It should also be mentioned that compounds containing arylsulfonyl radicals having nonhydrocarbon substituents can be used in the process and the corresponding intermediate and final products prepared, provided only that the substituent is non-reactive under the reaction conditions. Such non-reactive substituents include chlorine, bromine, and the methoxy, phenoxy, nitro and similar radicals. It should be mentioned further that, although the present invention is concerned primarily with compounds wherein the sulfonyl derivative is an arylsulfonyl derivative, the process can also be carried out and the corresponding intermediate and final products prepared using starting materials containing alkylsulfonyl, aralkylsulfonyl or cycloalkylsulfonyl radicals, such as the methane- .sulfonyl, ethanesulfonyl, cyclohexylsulfonyl, and

phenylmethanesulfonyl radicals.

The reaction of a dialkyl acetal oi 2,3-oxidopropanal with an N-(arylsulionyD-p-aminobenzoate ester can be carried out readily by heating the substances together, preferably with the addition of a catalytic proportion of pyridine or other tertiary amine. Approximately equimolecular proportions of the reactants can be used and the mixture preferably heated at from 'benzoate compoundcan be carried out conveniently by dissolving the ihydroxy compound and -chromic anhydride in glacial acetic acid and agitating the mixture for several hours, e. g. at

.generally consists of a mixture of liquid and solid in partially crystalline form upon allowing the F 'a'ction'mixture, the acid is precipitated in the form of a solid or oily-material which is generally somewhat yellowish in color.

about 100 to about 150" C. for from about 5 to about 60 minutes. The crude reaction mixture can be used directly in the subsequent oxidation-step of the process, if desired, or the N (3,3 dialkoxy 2 -'hydroxypropyl) N (arylsulfonyl) p aminobenzoate compound formed can be isolated from the reaction mixture, e. g. by washingthe crude reaction product thoroughly with a suitable solvent, such'as naphtha or'isopropanol, to remove the catalyst'and l0 unreacted starting materials. Some of the N (3,3- dialkoxy 2 --'hydroxypropyl) -"N (arylsulfonyl)-p-aminobenzoate esters are thus "obtained as crystalline compounds having well defined melting points and some as "sirupy 'liq- 15 uids. They can be "hydrolyzed to the'corresponding acids by mixing with aqueous alkalies "or other conventional hydrolytic agents and acidifying the aqueous-reaction mixture, preferably with a mineral-acid. Certain of the acids are thus obtained as solid substances.

The oxidation of an N-(3',3-dialkoxy-2 -hy- -droxypropyl) N (arylsulfonyl) p amino- '-benzoate compound to an N-(3,3-dialkoxy-2- ketopropyl) N (arylsulfonyl) p aminoroom temperature. The reaction mixture then substances from which the ketopropyl compound can be recovered in any convenient manner. If desired, the mixture can be mixed thoroughly with ether and filtered or it can be diluted with water andthen extracted with benzene or ethyl acetate. In either case, the organic layer or solutioncan'be washed'free of acid and inorganic salts with water, dried with sodium sulfate or other suitable agent andthe solvent then distilled in vacuo to leave the N-(3,3-dialkoxy 2 ketopropyl) N (arylsulfonyl) p aminobenzoate compound as a. sirupy' residue which is generally yellowish in color.

In some instances, the product can be obtained oily product, to stand. The crudeproduct can, however, be usedwithout furtherpurification in subsequent reactions. Gentle alkaline hydrolysis of the N- (3,3-dialkoxy-2-ketopropyl) -N-(arylsulfonyD-p-aminobenzoate compounds which are esters'leads to the formation of the corresponding acids. The hydrolysis canbe effected by mixing the ester with an aqueous or alcoholic alkali. Upon acidifying the cold aqueous re- The solid compounds can be purified, if desired, by recrystallization from alcohol.

The condensation of an. N- (3,3adialkoxy-2-kewarmed in aqueous 'orin glacial acetic acidsolu- 7 tion, e. g. at from room temperature toabout- "120C. for from afew minutes to a few hours.

The reaction product can be recovered -by-distilling the solvent or bydilutingthe mixture with water and separating the insoluble material. The product obtained is in the form of a dry powder which appears .to be substantially noncrystalline in character. 1 Reaction products which are esters can be hydrolyzed readily 'to the corresponding acids and, as noted previously. the arylsulfonyl radical can be-removed 'froln the molecule with hydrogen bromide.

The N (3,3 dialkoxy-2.-hydroxypropyl)eN- (arylsulfonyl) -p-aminobenzoate compounds and the N (3,3-dialkoxy-2-ketopropyl) -N-( ary1su1- fonyl)-p-aminobenzoate compounds having the formulae ,(III) and (IV), respectively, given in the reaction chart have, not been describedJpreviously. They can be. represented by thegeneric formula e COOR' aryl-SO: wherein Y is from the group consisting .of radicals having'the formulae -CHOH' and --CO-- and wherein n and R' havethe values given-previously. New compounds represented by the formula just given which can be prepared by the method herein described include, among many others, ethyl N (3,3 -'dimethoxy 2 hydroxypropyl) N (benzenesulfonyl) p aminobenzoate, methyl N-(3,3-diethoxy-2-ketopropyl) -N- (p-toluenesulfonyl) -p-aminobenzoate. n hexyl 'N (3,3 di-n-propoxy-Z-hydroxypropyl) N-(otoluenesulfonyl) -p-aminobenzoate, iso-propyl N- (3,3 di n hexoxy Z-ketopropyl) -N-(alphanaphthalenesulfonyl) -p-.aminobenzoate, n-butyl N (3,3 di n-butoxy-2-hydroxypropyl) N-;(pchlorobenzenesulfonyl) p aminobenzoate, noctyl N (3,3 di-n-octoxy-2-ketopropyl)-N-(p- .ethoxybenzenesulfonyl) p aminobenzoate, diethyl N'-(N- (3,3-dimethoxy-2-hydroxypropyl),- N (benzene-sulfonyl) p aminobenzoyll glutamate, dimethyl N'-(N-(3,3-diethoxy-2-ketopropyl) N (p-toluenesulfonyl) -p-(-aminobenvzoyl) glutamate, di n-hexyl .N--(N-(3, 3'-.di- ;npropoxy 2 hydroxypropyl) N (o-toluenesulfonyl) p .aminobenzoyl) glutamate, .di-isopropyl N' .(N-(3,3-di-n-hexoxy@2-ketopropy1)- 'N (alphanaphthalenesulfonyl). -Vp a aminobenzoyl) --.glutam ate, di-n-butyl N'-.(N-(3,3-d.i-iso.- butoxy 2 hydroxypropyl) -'N (p-bromobenzenesulfonyl) p aminobenzoyl) glutamate, di n octyl N (N-(3,3edi-n-nonoxye2-keto.- .propyl) N (o chloro p-ethoxybenzenesul- .fonyl) p-aminobenzoyl) glutamate, N-(3,3- dimethoxy -.2 hydroxypropyl) N-(benzenesulfonyl) p aminobenzoic acid, N-.(3,3-diethoxy- 2 ketopropyl) -N-(p-toluenesulfonyl)ep-aminobenzoic acid, N .(3,3-di-n-propoxy 2-hydroxypropyl) N (o toluenesulfonyl) peaminoben- ,zoic acid, N-(3,3-di-n-hexoxy-2sketopropyl) -.N-

(alpha naphthalenesulfonyl) peaminobenzoic acid, N (3,3-di-n-butoxy-2-hydroxypropyl)- Nr- (p-chlorobenzenesulfonyl)-p-aminobenzoic acid, N (3,3. di n octoxy 2- ketopropyl). -N-,(pw ethoxybenzenesulfonyl) p aminobenzdic acid, N' '(N (3,3-dimethoxy-Z-hydroxypropyl)+N- (benzenesulfonyl) p aminobenzoyD-glutamic acid, N (N-(3,3-diethoxy-2-ketopropyl) N-(ptoluenesulfonyl) p aminobenzoyl) glutamic acid, N (N (3,3 di n-propoxy-2-hydrdxypropyl) N (o --toluenesulfonyl) -;p aminobenzoyl) glutamic" acid, N'- (N- (3,3-jdi-n' hexoxy 2 'ketopropylr- --N (alpha -'-'naphthalenesul-'- fonyl) p aminobenzoyl) glutamic acid, N'-

-(N (3,3 di iso-butoxy-2-hydroxypropyl)-N- (p bromobenzenesulfonyl) p aminobenzoyl) glutamic acid, and N'-(N-(3,3-di-n-nonoxy-2- ketopropyl) N (o chloro-p-ethoxybenzenesulfonyl) -p-aminobenzoyl) -glutamic acid.

Certain advantages, in the invention are apparent from the following examples which are given by way of illustration only and are not to be construed as limiting.

Example 1.Ethyl N-(3,3-diethowy-2-hydroxypropyl) N-(p-toluenesuljonyl) -p-aminoben- .zoate A mixture of one gram of the diethyl acetal of 2,3-oxidopropanal, 1.5 grams of ethyl N-(p-toluenesulfonyl)-p-aminobenzoate and two drops of pyridine was heated for twelve minutes at l30135 C. The clear melt which was formed was cooled and seeded with crystals of previously prepared ethyl N-(3,3-diethoxy-2-hydroxypropyl) N (p-toluenesulfonyl)-p-aminobenzoate. The partially crystalline mass was triburated with a mixture of six milliliters of petroleum naphtha and three milliliters of isopropanol and the mixture filtered. There was thus obtained 1.71 grams of ethyl N-(3,3-diethoxy-2-hydroxypropyl) N (p-toluenesulfonyl)-p-aminobenzoate melting at 89-94 C. Repeated recrystallization of the product from isopropanol-petroleum naphtha raised the melting point to 91- 94 C. Hydrolysis of the ester with dilute alkali yields N- (3,3-diethoxy-2-hydroxypropyl) -N- (ptoluenesulfonyl) -paminobenzoic acid.

Emample 2.Ethyl N (3,3 diethoacy 2 ketcpropyl) N (p-toZuenesuljong Z) p aminobenzoate Approximately 3.2 grams of ethyl N-(p-toluenesulfonyl)-p-aminobenzoate was reacted with the diethyl acetal of 2,3-oxidopropanal by the method described in Example 1. The crude reaction mixture prior to seeding was dissolved in milliliters of glacial acetic acid and the solution mixed with a solution of one gram of chromic anhydride in 20 milliliters of glacial acetic acid. The mixture was allowed to stand at room temperature for about three hours and the acetic acid distilled in vacuo. The residue was triturated with ether and the mixture filtered. The ethereal filtrate was washed with water, dried and the ether volatilized. The residue consisted of ethyl N-(3,3-diethoxy-2-ketopropyl) N (p-toluenesulfonyl) p aminobenzoate in the form of an almost colorless oil.

N (3,3 diethoxy 2 ketopropyl) N (ptoluenesulfonyl)-p-aminobenzoic acid is prepared by hydrolyzing its ethyl ester or by using N (3,3 diethoxy 2 hydroxypropyl) N- '(p toluenesulfonyl) p aminobenzoic acid instead of its ethyl ester in the procedure just described.

Emample 3.Ethyt N-((2-amino-4-hydromy-6- 'pteridyDmethyZ) N (p toluenesulfonyllp-aminobenzoate A solution consisting of 100 milligrams of ethyl N (3,3 diethoxy 2 ketopropyl) N (ptoluenesulfonyl)-p-aminobenzoate, 296 milligrams of 2,4,5-triamino-6-hydroxypyrimidine. l milliliter ofwater and 2 millilitersor ethanol was 'adjustedto pH 4.0 and refluxed for '7 minutes. -The precipitate which formed was recovered by filtering and washing successively with water, alcohol and ether. The product thus recovered contained ethyl N-((2-amino-4-hydroxy-6-pteridyDmethyl) N (p toluenesulfonyl) paminobenzoate. The dry product was mixed with a solution of 1 gram of sodium hydroxide in 2 milliliters of water and the mixture allowed to stand for 16 hours at room temperature. The pH of the mixture was then adjusted to 3.2 with mineral acid and the insoluble material collected by centrifuging and washed with water. There was thus obtained 25 milligrams of N-( (2-amino- 4 hydroxy 6 pteridyDmethyl) N. p-toluenesulfonyl) -p-aminobenzoic acid.

Example 4.Hydrolysis and cleavage of ethyl N- ((2 amino 4 hydroxy 6 pterz'dylimethz l) N (p toluenesuljonyl) p aminobenzoate The crude ethyl N-((2-amino-4-hydroxy-6- pteridyDmethyl) N (p toluenesulfonyl) paminobenzoate was dissolved in 3.5 milliliters of a 26 per cent by weight solution of hydrogen bromide in glacial acetic acid and 0.13 gram of phenol was added quickly. The mixture was stirred at room temperature for one hour and then poured into 30 milliliters of anhydrous ether. After mixing thoroughly and allowing to stand for a short time, the mixture of ether and suspended solid material was filtered and the residue washed twice with anhydrous ether and then dried in vacuo for several hours. The dry resi due consisted of impure ethyl N-((2-amino-4 hydroxy 6 pteridyl) methyl) p aminobenzoate. The product was hydrolyzed with dilute sodium hydroxide to convert the ester to the free acid. The product had an activity when bio-assayed with Streptococcus iecalis R corresponding to a purity of 32 per cent of N-((2- amino 4 hydroxy 6 pteridyl) methyl) paminobenzoic acid.

Example 5.Diethyl N (N-(p-toluenesulfonyl) p-aminobenzoyl) -1-glutamate Thirty and nine-tenths grams of N-(p-toluenesulfonyD-p-aminobenzoyl chloride and 23.9 grams of diethyl l(+)-glutamate hydrochloride were dissolved in 300 milliliters of ethylene dichloride and the solution cooled to between 0 and 10 C. The cold solution was stirred vigorously and 22.3 grams of triethylamine in 72 milliliters of ethylene dichloride was added slowly over a period of about 20 minutes. The temperature of the mixture was held between 10 and 20 C. during the addition of the triethylamine and the mixture then allowed to stand at room temperature for one hour. The mixture was then washed successively with water, dilute hydrochloric acid, saturated aqueous sodium bicarbonate and finally with water. The colorless solution thus obtained was dried with anhydrous sodium sulfate and naphtha was added until the solution became opalescent. The mixture was then cooled to cause crystallization and filtered. The crystals, after drying, consisted of 36 grams of diethyl N'-(N-(p-to1uenesulfonyl) p aminobenzoyl) 1 glutamate melting at 124 to 126 C.

ldroxypropyl) N a (p --toluenesulfony,l) 12- aminobenzoyl) -1 -glutamate One and six-tenths grams of the diethyl acetal of 2.3-oxidopropanal and five drops of pyridine were added to 4.77 grams of fused diethyl N'- (N-- (ptoluenesulionyl) p aminobenzoybl-glutamate at 140 C. The mixture was stirred for about 30 minutes at 145 to 150 C. The highly colored mass consisted chiefly of diethyl N (N (3,3 diethoxy 2 hydroxypropyD- N (p toluenesulfonyl) p aminobenzoyD- l-glutamate. It had an index of refraction without further purification of N =1.527. N-(N- (3,3 diethoxy 2 hydroxypropyly- N (ptoluenesulfonyl) p aminobenzoyl) 1 glutamic acid is formed by hydrolyzing the ester with dilute alkali.

Example 7.Diethyl N (N (3,3 diethoxy-Z- ketopropyl) N (p toluenesulfonyl) paminobenzoyl) -1 -glutamate Five grams of diethyl N"-(N-(p.-toluenesulfonyl) p aminobenzoyl) -1-glutamate and 1.9 grams of the diethyl acetal of 2,3-oxidopropanal were reacted substantially as by the method of Example 6. The crude diethyl N"-(N-(3,3-diethoxy 2 hydroxypropyD-N-(p -toluenesulfonyl)-p-amin-obenzoyl)-1-glutamate thus obtained was dissolved in 35 milliliters of glacial acetic acid and 1.7 grams of chromic anhydride was added to the solution. The mixture was stirred at room temperature for about two hours andthen filtered. The filtratewas diluted with water and extracted with a mixture of benzene and ethyl acetate. The organic layer was separated, washed free of acid with water and dried over'anhydrous sodium sulfate. The solvent wasthen distilled in vacuo and there was thus obtained 5.9 grams of diethyl N'-(N- (3,3-diethoxy- 2 ketopropyl) -N-(p-toluenesulfonyl) -p-aminobenzoyl) 1-g1utamate as a yellow 'sirup. Hydrolysis of the ester with dilute-alcoholic sodium hydroxide forms N'-(N-(3,3-diethoxy-2-ketopropyl)-N-(p-toluenesulfony1) p aminobenzoyl) -1-glutamate acid.

Er'cample; 8'.--Dieth'yl N (N -:(2.';-amino;.- 41 ml dfoxy 6'- pt'eridyDmetfiyD-N- (p toluenesulfonyl) -p-amznobenzoyl) -1-glutamat of diethyl N'-(N-(2-amino 4.- hydroxy-fi-ptera idyDmethyI-N-(p-toluenesulfonyl). p amino-.

benzoyl) -1-glutamate. N'(N (2 aminor l-hydroxy 6 pteri'dybmethyl N- (p toluenesulfonyl) p aminobenzoyl) 1 glutamic acid'is formed in similar manner using N*-(N-(3,3-diethoxy-2-ketopropyl) N (p toluencsulfonyl) HI I p-aminobenzoyb l-glutamic acid instead of? its diethyl ester in-theprocedure just described.

Ewample 9.Hydrolysz's and cleavage of diethyl N'(N-((2. amino 4 hydroxy 6 pteridyl) methyl) N (p-toluenesulfonyl) p aminobenzoyl) -glutamate The crude reaction product containing diethyl N"-(N ((2 amino 4 hydroxy 6 pteridyl) methyD-N-(p toluenesulfonyl p aminobenzoyl)glutamate obtained in Example 8 was mixed with milligrams of phenol and 2.5. milliliters of a 26 per cent solution of hydrogen bromide in glacial acetic acid. The mixture was stirred for ninety minutes at room temperature and then diluted with anhydrous ether; Ethylpteroyl glutamate hydrobromide separated from the mixture as a, brown, hygroscopic solidwhich was recovered by filtration. The brown solid was dissolved in about two milliliters of methanol and ten per cent aqueous sodium hydroxide was added to give a strongly alkaline solution. The alkaline mixture. was allowed to stand for ninety minutes at 40 C. andthen acidified with mineral acid to pH 3.0. The solution was then diluted with five milliliters of water, cooled in an ice bath for one hour and then centrifuged. The solid residue was washed with three milliliters of water and. three times with. three milliliters of acetone and dried in vacuo over sulfuric acid. There was thus obtained 89 milligrams of pteroyl glutamic acid Which, upon microbiological assay with Lactobacillus casei had an activity of 48.8 per cent of that of the pure natural acid.

We claim:

1. The process of preparing N-((2-amino-4- hydroxy 6 pteri-dyl) methyl) -N- (arylsulfonyl) p-aminobenzoate compounds which includes the step of reacting a keto acetal having the formula wherein R is, from the group consisting or hydrogen and the alkyl radicals and nis fromtligroup consisting of zero and" the positive integer- 1, with 2,4,5-triamino=6-hydroxypyrimidine to form a compound having the formula on A N 000R! N oHrN-Ooomndnomomomnon' wherein R and n have the valuesgiven;

2. The method (if-claimv 1 wherein n is zero. 3. The method of claim 1 wherein n. is the positive integer, 1.

4. The method of claim 1 wherein R is hydrogen.

5. The method of claim 1 wherein R is an alkyl radical.

6. The method of claim 1 wherein the alkyl radicals each contains less than 8 carbon atoms.

'7. The method of claim 1 whereinthe-arylsul fonyl radical is the p-toluenesulfonyl radical.

8. The method of claim. 1 wherein the keto acetal is ethylN- (3,3 -diethoxy-2-ketopropyl) N- ('p-tolenesulfonyl) -p-aminobenz'oate;

9. The methodoiclaim 1 wherein the keto acetal is diethyl N-(N-(3,3-diethoxy=2rketo= propyl) N- (p toluenesulfonyl) -p-aminobenzoyl) -glutamate.

11 10; The method which includes: oxidizing an hydroxy acetal having the formula C OR 0 C(NHCHCHzGHaC O)IIOR' alkyl ester of N (N (p toluenesulfonyl) -p-'" aminobenzoyl) -glutamic acid.

14. The method which includes: reacting a dialkyl acetal of 1,2-oxidopropanal with an ester 0 o 0 R (e.lky1O)zCHC 0CHi-N comnonomomcoinoh' aryl-SOz at; insistfsnsitsttszzisri hmngformuia a comnound having the formula C 0 0 a1ky1 000R, HN CO(NH( JHOH1CHiC0).0elkyl aryl-SO:

N\ on N-OCO J: I (NH nomomoohon HzNkN R and n having the values given.

wherein n is from the group consisting of zero and the positive integer 1, to form an hydroxy acetal ester having the formula GOO-alkyl 11. The method which includes: reacting a diaryl-SO: hydrolyzing the hydroxy acetal ester with an alkali to form an hydroxy acetal acid having the formula COOH alkyl acetal of 1,2-oxidopropanal with an ester aryl- O2 oxidizing the hydroxy acetal acid to form a keto acetal acid having the formula coon having the formula c 0 O-alkYl wherein n is from the group consisting of zero and the positive integer 1, to form an hydroxy acetal ester having the formula 71. having the valuegiven.

co O-ulkyl aik l-o-non-cnon-cnr-l Ooomnorromomoo)..o-a1k 1 aryl-SO; oxidizing the hydroxy acetal ester to form a keto acetal ester having the formula Y c o 0-alkyl 15. The process according to claim 1, wherein the reactants are heated together in the pres- (alkyl-OhCH-O O-CHa-NQCO(NHHCHzCHzCOhO-alky I ary1-S02 and reacting the keto ester acetal with 2,4,5- triamino-6-hydroxy-pyrimidine to form a compound having the formula COO-alkyl at having the value given.

12. The method of claim 11 wherein a dialkyl acetal of 1,-2-oxidopropanal is reacted with an alkyl ester of N-(p-toluenesulfonyl) -p-amino- 'benzoic acid.

13. The method of claim 11 wherein a dialkyl acetal of 1,2-oxidopropanal is reacted with a di- 7t ence of a solvent at a temperature below about 0 degrees centigrade.

DAVID I. WEISBLAT. BARNEY J. MAGERLEHI.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,211,467 Kimball et al Aug. 13, 1940 2,444,002 Boothe June 22, 1948 2,444,005 Cosulich June 22, 1948 2,465,316 Mowry Mar. 22, 1949 OTHER REFERENCES Lederle Bulletin, 13, (No. 3) 21, (1948). 

14. THE METHOD WHICH INCLUDES: REACTING A DIALKYL ACETAL OF 1-2-OXIDOPROPANAL WITH AN ESTER HAVING THE FORMULA 